The Wnt pathway is an evolutionarily conserved signaling pathway present in metazoans from Drosophila to humans. Wnt signaling has been shown to play important roles in development. Given that the Wnt pathway is involved in the genesis of a wide variety of human diseases (e.g., over 90% of all colorectal cancers), there is an intense effort to develop therapeutics that target this pathway. Unfortunately, in part due to our incomplete understanding of the detailed mechanism of Wnt signal transduction, progress in developing therapeutics that target this pathway has been slow, and no Wnt inhibitors are currently in clinical use. Our overarching goal is to understand the basic biochemical mechanisms by which a Wnt signal is propagated to ultimately coordinate the formation of tissues, organs, and limbs and to understand how its misregulation can lead to disease states. In over more than a decade, my laboratory has 1) developed the first biochemical system (Xenopus egg extract) that recapitulated key reactions of the Wnt pathway, 2) developed the first mathematical model (Lee- Heinrich model) of the Wnt pathway, 3) provided evidence for a mechanism involving receptor-mediated signaling (via direct inhibition of GSK3 activity) to the cytoplasm, and 4) identified a small molecule inhibitor of the Wnt pathway that has been designated by the FDA as an orphan drug for a familial precancerous disease (familial adenomatous polyposis). Our recently funded work focuses on 1) our newly proposed model describing how ?-catenin, a Wnt transcriptional coactivator, competes with the corepressor Gro/TLE for binding to the transcriptional factor, TCF/LEF, via a process facilitated by the E3 ligase XIAP and 2) a newly identified mechanism by which loss of the APC tumor suppressor leads to activation of Wnt cell surface receptors. For the MIRA grant application, I propose to extend these studies by 1) determining the molecular mechanism by which APC regulates receptor activation, specifically the role of vesicle transport in this process, and 2) pursuing the characterization of a deubiqutinase, USP47, which interacts with XIAP to promote ?-catenin-mediated Wnt signaling. We have undertaken several genome-scale screens to identify new Wnt pathway genes. Based on the results of these screens, I propose to study 1) Wnt signalosome formation involving the transmembrane and vesicular transport proteins: Arl4c, ITSN1, Syndecan-2, and Cdh13, 2) Wnt receptor homeostasis by the USP46/UAF1/WDR20 deubiquitinase complex, and 3) regulation of Wnt gene transcription by the nuclear kinase STK38.